This application is related to Japanese Patent Application No. 2000-184853 filed on Jun. 20, 2000, whose priority is claimed under 35 USC xc2xa7119, the disclosure of which is incorporated by reference in its entirety.
1. Field of the Invention
The present invention relates to a method for quantitatively analyzing fragmented red blood cells (FRCs) and, particularly, to a method for quantitatively analyzing fragmented red blood cells observed in peripheral blood in the case of various diseases such as cardiovascular abnormality, congenital or acquired hemolytic anemia, disseminated intravascular coagulation, hemolytic uremic syndrome and TTP (Thrombotic Thrombocytopenic Purpura).
2. Description of the Related Art
With recent studies on BMT-TMA (bone marrow transplantation-associated thrombotic microangiopathy), attention has been given to FRC % (the percentage of fragmented red blood cells) in peripheral blood, which is regarded as an important index for early diagnosis of BMT-TMA. A BMT-TMA grading system (grades of 0 to 4) was developed on the basis of the FRC % as well as the lactate dehydrogenase (LDH) (Bone Marrow Transplantation, 15, p. 247-253, 1995). The grades are defined as follows:
Grade 0 Normal LDH and FRCxe2x89xa61.2%
Grade 1 Normal LDH and FRCxe2x89xa71.3%;
Grade 2 Increased LDH and FRC=1.3% to 4.8%;
Grade 3 Increased LDH and ERG=4.9% to 9.6%; and
Grade 4 Increased LDH and ERGxe2x89xa79.7%
The FRC % can be determined through visual observation of a peripheral blood smear film. In general, the FRCs are not quantitatively analyzed, but analyzed for detection thereof. Although there are several reports which state that evidence of FRCs can be detected on the basis of a red blood cell size distribution standardized for automatic red blood cell counters (American Journal of Clinical Pathology, 90, p. 268-273, 1988), the quantitative analysis of the ERCs still relies on the visual observation of a smear film, and the results of the observation may vary significantly with different observers due to absence of criteria to be referenced.
In view of the foregoing, the present invention is directed to a method for quantitatively analyzing fragmented red blood cells, which can determine the FRC % with a high level of accuracy by establishing a specific area on a scattergram of a flow cytometer.
According to the present invention, there is provided a method for quantitatively analyzing fragmented red blood cells, comprising the steps of: adding a nucleic acid staining fluorochrome dye to a blood sample for preparation of a specimen containing particles; allowing the specimen to flow through a flow cytometer, and measuring a scattered light intensity and a fluorescent light intensity for each particle in the specimen; preparing a two-dimensional particle distribution diagram on the basis of the measured scattered light intensity and fluorescent light intensity; establishing a red blood cell distribution area on the two-dimensional distribution diagram, and counting particles falling within the red blood cell distribution area for determination of the total number A of red blood cells; establishing a fragmented red blood cell distribution area in a region in which the scattered light intensity and the fluorescent light intensity are low within the red blood cell distribution area, and counting particles falling within the fragmented red blood cell distribution area for determination of the number B of fragmented red blood cells; and calculating a value B/A, and calculating a content by percentage F of the fragmented red blood cells from a conversion function F=f(B/A).